Targeting gene delivery to activated vascular endothelium using anti E/P-Selectin antibody linked to PAMAM dendrimers

Introduction

The adhesion molecules P- and E-selectin are expressed on activated endothelial cells, and are good targets for gene therapy aimed at inflammatory disease. We have therefore investigated the potential of targeting PAMAM dendrimers, a non viral vector system, to cells expressing P/E-selectin using a monoclonal antibody that recognises these molecules.

Materials and Methods

We used biotin and avidin to cross-link anti E/P-Selectin monoclonal antibody to pre-formed Superfect-DNA complexes that were then used to transfect reporter genes to CHO cells expressing E-Selectin, cytokine-activated primary Human Saphenous Vein Endothelial Cells (HSVEC) and whole vein segments.

Results

The use of the anti E/P-Selectin antibody increased the transfection efficiency in CHO-E cells, activated HSVEC and saphenous vein segments ex vivo. We also showed that the antibody improved the binding of the complexes onto cells as well as the internalisation kinetics.

Discussion

We demonstrate here that by attaching antibodies onto PAMAM dendrimers the efficiency of transfection can be significantly improved in cells or tissues expressing the receptor. This technology has potential in the treatment of cardiovascular disease by gene therapy but can also be used with different antibodies to target other diseased cells or tissues.     

鸟苷酰环化酶C小干扰RNA对胃癌细胞生长的影响

目的：研究靶向鸟苷酰环化酶C（Guanylyl cyclaseC,GC-C）的小干扰RNA（siRNA）对人胃癌SGC-7901细胞体外生长能力的影响。方法：利用pSilencer TM4.1-CMVneo通过siRNA表达载体法制备GC-CsiRNA。将GC-CsiRNA转染胃癌SGC-7901细胞,采用RT-PCR和间接免疫荧光观察转染前后胃癌细胞GC-C基因的表达变化,黏附实验观察转染前后胃癌细胞黏附能力。结果：构建的GC-CsiRNA测序鉴定与设计完全相符。GC-CsiRNA转染胃癌SGC-7901细胞后,明显抑制GC-C基因的表达。转染后,GC-CmRNA和蛋白表达分别抑制了66.7%和75.8%,细胞黏附能力较对照组减弱（P〈0.05）。结论：靶向GC-C的siRNA可明显下调靶基因GC-C的表达,在体外抑制胃癌SGC-7901细胞的黏附,提示GC-C基因与胃癌的发生、发展有密切的关系。     

Cutaneous lymphocyte-associated antigen (CLA) T cells up-regulate P-selectin ligand expression upon their activation

Memory T cells expressing CLA occur in humans and accumulate in normal and inflamed skin. These cells uniformly bind to the vascular adhesion molecule E-selectin, yet only a subset binds to P-selectin. The latter cells are distinguished by the mAb CHO-131, and are enriched in psoriasis lesions. Activated T cells up-regulate CLA expression, but little is currently known about their binding to P-selectin. We observed that CLA⁺ CD4⁺ T cells derived from stimulated naive T cells uniformly express the CHO-131 epitope. This occurred as well upon the restimulation of memory CLA⁺ CD4⁺ T cells. The latter cells also expressed higher levels of PSGL-1 modified by P-selectin glycan ligands; C2GlcNAcT-1 mRNA, a glycosyltransferase critical for such glycan synthesis; and more uniformly bound to P-selectin. Our findings thus indicate that unlike memory CLA⁺ CD4⁺ T cells, when activated these cells can broadly bind to P-selectin, suggesting a more diverse tissue trafficking capacity.     

Decursinol angelate blocks transmigration and inflammatory activation of cancer cells through inhibition of PI3K,ERK and NF-κB activation

Inflammation is known to be closely associated with the development of cancer. Decursinol angelate (DA), a coumarin compound isolated from Angelica gigas and related compounds have been shown to possess potent anti-inflammatory activities. However, little is known about their effects on the inflammatory processes associated with cancer. In this study, the anti-inflammatory effect of DA was evaluated in cancer cell lines with respect to cellular invasion through the extracellular matrix (ECM) and the expression of pro-inflammatory mediators such as cytokine, cell adhesion molecules and matrix metalloproteinase (MMP)-9. DA inhibited the invasion of fibrosarcoma cell line, HT1080 and breast cancer cell line, MDA-MB-231 in the Matrigel invasion assay. DA-mediated suppression of cancer cell invasion was accomplished by suppression of PI3K activity known to be associated with cytoskeletal rearrangement related to cellular migration. DA also suppressed the adhesion of cancer cells to ECM mediated by down-regulation of β₁-integrin expression levels. Furthermore, DA inhibited the expression of pro-inflammatory cytokines and MMP-9 through suppression of PI3K, ERK and NF-κB activation. These results demonstrate that DA suppresses invasion and inflammatory activation of cancer cells through modulation of PI3K/AKT, ERK and NF-κB. These anti-inflammatory activities of DA may contribute to its anti-cancer activity.     

LKB1 Regulated Pathways in Lung Cancer Invasion and Metastasis

Metastasis is characterized by the ability of cancer cells to invade into adjacent tissue, intravasate into blood or lymphatic vessels, and extravasate into a distant tissue. Metastatic disease is primarily responsible for the low 5-year survival rate of non-small cell lung cancer (NSCLC), and therefore, an understanding of the molecular mechanisms that regulate NSCLC metastasis is clearly warranted. The serine/threonine kinase and tumor suppressor LKB1 is mutated in 30% of NSCLC tumors, and recent evidence points to a prominent role in NSCLC metastasis. This review summarizes LKB1-dependent invasion pathways where compromised LKB1 function could promote NSCLC metastasis.     

原发性IgA肾病患儿肾组织免疫病理及CD44的表达

目的:探讨细胞表面糖蛋白(CD44)在儿童IgA肾病(IgAN)发病中的作用及其与肾小管间质损害的关系。方法:46例IgAN患儿按临床表现分为3组:孤立性血尿组14例,血尿蛋白尿组17例,肾病综合征组15例;按病理分级分为3组:病理Ⅰ+Ⅱ级组23例,Ⅲ级组14例,Ⅳ级组9例。设肾活检正常对照组4例。采用免疫组化SP法检测病例组与对照组肾组织CD44的表达,并分析其与肾小管间质病变程度的相关性。结果:①正常肾组织仅有少量CD44表达。②IgAN患儿肾组织中CD44的表达均较对照组明显增高,不同临床表现组中肾病综合征组最高,孤立性血尿组最低,且与IgAN患儿的病理分级密切相关。③IgAN患儿肾组织CD44的表达与肾小管间质损害程度呈正相关。结论:CD44参与了儿童IgAN的发病和肾小管间质损害,与IgAN的进展及预后有关。     

粘附分子CD62P和CD44在毛细支气管炎患儿外周血中的表达及意义

目的 探讨黏附分子CD62P和CD44在毛细支气管炎(简称毛支炎)患儿外周血中的表达及意义。方法 选取2014 年11 月至2015 年5 月住院治疗的毛支炎发病期患儿33例和恢复期患儿19例为研究对象,同期选取支气管肺炎患儿30例为支气管肺炎组,非感染患儿26例为对照组。采用流式细胞术检测各组患儿外周血CD62P的表达百分比,ELISA法测定血清CD44的水平。结果 毛支炎发病期组CD62P和CD44水平显著高于毛支炎恢复期组、支气管肺炎组及对照组(PPr=0.91,P结论 黏附分子CD62P、CD44参与了毛细支气管炎的发病过程,其水平高低可反映毛细支气管炎炎症反应的严重程度。     

Decrease in circulating endothelial cell adhesion molecule and thrombomodulin levels during oral iloprost treatment in rheumatoid arthritis patients: preliminary results

Objective: Rheumatoid arthritis is a chronic inflammatory autoimmune disease with proinflammatory cytokines involved in its pathogenesis. Recently in vitro as well as in vivo studies have shown that iloprost, a stable prostacyclin analogue, can reduce the release of these cytokines. This study was performed to further investigate the anti-inflammatory effects of iloprost by determining plasma adhesion molecules as markers of endothelial cell activation, and plasma thrombomodulin as a parameter of endothelial cell injury in patients with rheumatoid arthritis receiving oral iloprost therapy. Methods: Plasma thrombomodulin levels and the values of the plasma adhesion molecules VCAM-1 (vascular cell adhesion molecule 1), E-selectin (CD62E), and ICAM-1 (intercellular adhesion molecule 1, CD 54) were measured by ELISA during a 7-day period of treatment with orally-administered iloprost in 14 patients with active rheumatoid arthritis. Finally, the same parameters were determined at the end of the observation period (1 week after the end of therapy). In addition, the disease activity was measured using the DAS (disease activity score) as well as the patients self-assessed pain severity, and correlated with the changes of plasma adhesion molecule and thrombomodulin levels. Results The plasma levels of all three adhesion molecules as well as of thrombomodulin significantly decreased under therapy with oral iloprost. After 1 week (day 7 of therapy), the mean percent changes from day 0 were –20.1% for VCAM-1 (p=0.008), –21.2 for ICAM-1 (p=0.003), –24.6% for E-selectin (p=0.001), and –21.7% for thrombomodulin (p=0.003). This decrease lasted up to 1 week after the end of therapy in the case of VCAM-1 (p=0.023) and ICAM-1 (p=0.001). Further analysis of the results revealed additional significant correlations between different parameters of clinical disease activity, thrombomodulin and adhesion molecules. Conclusion This study showed hints towards clinical effects in patients with rheumatoid arthritis receiving oral iloprost therapy. Pathophysiologically, the decrease of adhesion molecules points at an immunomodulating effect of iloprost. The observed thrombomodulin-lowering effect of iloprost may indicate stabilisation of endothelial cell function by diminishing endothelial cell injury.     

Membrane mobility and clustering of Integrin Associated Protein (IAP, CD47)--major differences between mouse and man and implications for signaling

Integrin Associated Protein (IAP, CD47) is a ubiquitous integral membrane protein implicated in processes (in mice) that range from inhibiting clearance by phagocytes [Oldenborg et al., Science 2000; Gardai et al., Cell 2005] to neutrophil motility [Lindberg et al., Science 1996]. SIRPalpha is CD47's main receptor on phagocytes plus a number of other cell types, and SIRPalpha-CD47 interactions in clusters are believed to mediate signaling. However, considerable species differences in CD47 sequence as well as differences in CD47 extractability from mouse cells versus man motivate a characterization of mobility, clusterability, and kinetics under force of CD47-SIRPalpha. Despite similar levels of CD47 on red cells from mouse and man, we find an effective avidity of SIRPalpha-CD47 for mouse appears higher than for human. Both mouse and human CD47 show clustering by multivalent SIRPalpha complexes, but only mouse cells aggregate with CD47 concentrating at cell-cell contacts. This proves consistent with fluorescence imaged micro-deformation, which indicates near-complete mobility of CD47 on mouse cells compared to only about 30-40% mobility on normal human cells. To qualify the method, we also show that disrupting cellular F-actin dramatically increases the mobility of integral membrane proteins. Furthermore, atomic force microscopy probing of cell membranes with human SIRPalpha confirms the species-specific interactions and provides evidence of clustering and adhesion on short time scales, but it also shows surprisingly strong forces in detachment for a signaling complex. The results thus highlight major species differences in CD47-SIRPalpha interactions and CD47 integration, suggesting that signaling by CD47 in man may be qualitatively different from mouse.